DMT1 is a widely-expressed iron transporter that is essential for adequate intestinal absorption of iron and for transport of iron in red blood cell precursors for the production of hemoglobin. Rare mutations in DMT1 cause severe microcytic anemia. Conversely, dysregulation of DMT1 in hereditary hemochromatosis, the most common hereditary disease in Caucasians, results in toxic iron overload in vital organs. Since there exists no regulated mechanism for its excretion, iron homeostasis is achieved by regulating intestinal iron absorption. DMT1 is the gateway for iron absorption - it is the primary or only apical transporter of nonheme iron - making it the focus of this proposal. Iron deficiency affects as much as 10% of the U.S. population and is the most prevalent micronutrient deficiency worldwide. Important for this proposal, iron deficiency is a serious risk factor for cadmium intoxication, suggesting that cadmium and iron share a common absorptive mechanism. Under investigation in this proposal is the premise that DMT1 is a complex H+-coupled and voltage-dependent ferrous-iron (Fe2+) transporter and that an acidic microclimate at the intestinal brush-border membrane provides the proton-motive force energizing DMT1. We will probe the molecular basis of H+-coupling and explore a physiological role for the significant uncoupled H+ fluxes (slippage) through DMT1 using the voltage clamp, radiotracer (55Fe) assays, and fluorescence approaches in Xenopus oocytes expressing wildtype DMT1 or mutant proteins. Next, we will examine iron transport in the mouse intestine using radiotracer and fluorescence approaches; the use of specific inhibitors of intestinal Na+/H+ exchangers (NHE3 and NHE2) and mutant mouse models lacking NHE3, NHE2, or the gastric H+/K+-ATPase will permit us to evaluate the roles of gastric acid and the intestinal brush-border acidic microclimate in providing the H+ to drive DMT1-mediated Fe2+ transport. The second premise is that DMT1 serves absorption not only of iron but also of certain other essential metals such as Co and Mn (as well as trace metals such as Ni and V) but that this promiscuity also makes it a major route of entry for the toxic heavy metal Cd. Here, we will determine the comprehensive substrate profile and metal-ion selectivity of DMT1 using our oocyte assays. Next, we will use the Belgrade anemic rat (which bears a mutation in DMT1) to examine the physiological significance of DMT1 in the absorption of each of the metals it is capable of transporting. Finally, we will identify metal-coordination sites in DMT1. Studying DMT1 in this way will lead to a better understanding of the mechanisms of nonheme iron absorption, the conditions required for efficient absorption, and the role of DMT1 in the metabolism of other transition metals. The results of this work will help drive development of new strategies for improving metal nutrition, or for treating iron overload and heavy-metal intoxication. Public Health Relevance: Iron deficiency leads to anemia and the hereditary disease hemochromatosis leads to toxic iron overload, illustrating the importance of balancing intestinal iron absorption. The gateway for dietary iron to enter the cells lining the intestine is a protein called DMT1, which can also transport the toxic metal cadmium. In this project we will study iron and cadmium transport via DMT1 at the molecular level to drive development of new strategies to improve iron nutrition and prevent the toxic effects of cadmium or iron overload.